Photosynthesis
Plant leaves are the site of photosynthesis, a process by which the energy in sunlight is harnessed and used to generate foodstuffs. As sunlight is gathered, water is split, with its hydrogens used to reduce certain chemical compounds and its oxygen used to form O.sub.2. The reduction of chemical compounds by the light-driven mechanisms of photosynthesis is termed the "light reaction"; the use of these reduced compounds to fix CO.sub.2 into foodstuffs is termed the "dark reaction" (Taiz, L. and Zeiger, E., Plant Physiology, Benjamin/Cummings Pub. Co., Inc., Redwood City, Calif., 1991, Chap. 8-10, p. 177).
The light and dark "reactions" are not single steps, but instead comprise several complex series of reactions, each involving electron transfer (Taiz and Zeiger, 1991). The light reaction is divided into two "photosystems." Photosystem II (PS.sub.II) comprises the reactions leading to the splitting of water and the release of oxygen. Photosystem I (PS.sub.I) comprises the reactions leading to the reduction of NADP.sup.+ (nicotinamide adenine dinucleotide phosphate) to NADPH, a compound which provides energy used in the dark reaction (Taiz and Zeiger, 1991).
Plant Fluorescence
Chlorophylls and certain other molecules involved in photosynthesis absorb visible light, "funneling" the absorbed energy in the form of free electrons to reaction centers (RCs) that are parts of the two photosystems (Taiz and Zeiger, 1991; Bolhar-Nordenkampf, H. R. and Oquist, G., "Chlorophyll Fluorescence as a Tool in Photosynthesis Research," in Photosynthesis and Production in a Changing Environment, a Field and Laboratory Manual, Hall, D. O., Scurlock, J. M. O., Bolhar-Nordenkampf, H. R., Leegood, R. C. and Long, S. P. Eds., Chapman and Hall, London, 1993, Chap. 12, p. 193). When the RCs are maximally available (open), about 97% of the absorbed light energy is productively used for photochemical reactions leading to the production of foodstuffs, about 2.5% is "lost" as heat, and about 0.5% is re-emitted at longer wavelengths as fluorescence (Bolhar-Nordenkampf and Oquist, 1993). When the RCs are not available (closed), about 90-95% of absorbed light energy is lost as heat, and about 2.5-5% is re-emitted as fluorescence (Bolhar-Nordenkampf and Oquist, 1993).
Fluorescence from photosynthetic systems is thus tightly coupled to the availability of RCs, as is especially evident when dark-adapted systems are first illuminated. When photosynthetic systems are initially illuminated, fluorescence is low because most RCs are open. However, with continued illumination, fluorescence increases and then varies with time in a characteristic manner as the RCs are recruited into use and the light energy is employed to Dower various reactions (Bolhar-Nordenkampf and Oquist, 1993). If the illumination is constant, a plot of fluorescence versus time will have a characteristic shape known as the Kautsky curve (Kautsky, H. and Hirsch, A., Biochem. Zeitschrift 274, 423, 1934). Important features of this curve have been designated by the upper-case letters OIDPSMT, corresponding to Origin of curve or initial fluorescence, Initial rise, Dip, Peak, down Slope (or quasi-steady-state) between-primary (P) and secondary (M) maxima, and Terminal level, respectively (Lavorel, J. and Etienne, A. L., "In Vivo Chlorophyll Fluorescence," in Topics in Photosynthesis, 2, Barber, J. Ed., Elsevier, Amsterdam, 1977, p. 203).
OIDPSMT transients reflect the availability of RCs and the rate of electron flow through the electron transport chain of the light reaction (Mawson, B. T., Morrissey, P. J., Gomez, A. and Melis, A., Plant Cell Physiol. 35, 341, 1994; Papageorgiou, G., "Chlorophyll Fluorescence: an Intrinsic Probe of Photosynthesis," in Bioenergetics of Photosynthesis, Govindjee [no first initial] Ed., Academic Press, New York, 1975, p. 319.; Powles, S. B., Ann. Rev. Plant Physiol. 35:14, 1984; Edner, H., Johansson, J., Svanberg, S. and Wallinder, E., Applied Optics 33, 2471, 1994; Methy, M., Olioso, A. and Trabaud, L., Remote Sens. Environ. 47, 42, 1994). When a dark-adapted system is illuminated, chlorophyll antenna in light-harvesting complex (LHC) II and PS.sub.II quickly begin funneling light energy in the form of free electrons to the RCs of PS.sub.II, closing RCs and increasing fluorescence within picoseconds (transient O). However, free electrons within the RCs are transferred within milliseconds to electron acceptor Q.sub.A (transient I) and electron acceptor Q.sub.B (transient D), re-opening the RCs and decreasing the fluorescence. Q.sub.A and Q.sub.B are saturated within a millisecond, again closing RCs and increasing fluorescence, which reaches a maximum and peaks within about 0.5-2 seconds (transient P). A minimum (transient S) and second maximum (transient S) occur as electrons are transferred to LHCI and PS.sub.I, which then saturate. By this time, the dark reactions of photosynthesis begin demanding reducing "power," which re-opens the RCs and reduces fluorescence to background levels after a hundred to several hundred seconds (transient T).
Practical Implications of Plant Fluorescence
The time dependence of plant fluorescence manifest in the OIDPSMT curve can be used to detect pathological and physiological changes in plants and differences between plants. For example, the OIDPSMT curve depends on the physiological condition and type of plant, the structure and potential for activity of the photosynthetic apparatus, the demands of the dark reaction, the health of the leaf, and the plant's adaptation to environmental conditions (Tecsi, L. I., Maule, A. J., Smith, A. M and Leegood, R. C., Plant J. 5, 837, 1994; Havaux, M. and Lannoye, R., Photosynthetica 18, 117, 1984; Omasa, K., Shimazaki, K., Aiga, L., Larcher, W. and Onoe, M., Plant Physiol. 84, 748, 1987; Bolhar-Nordenkampf, H. R. and Lechner, E. G., "Winter Stress and Chlorophyll Fluorescence in Norway Spruce (Picea abies (L.) Karst)," in Applications of Chlorophyll Fluorescence, Lichtenthaler, H. K. Ed., Kluwer Academic Publ., Dordretcht, Holland, 1988, p. 173; Ghirardi, M. L. and Melis, A., Biochem. Biophys. Acta 932, 130, 1988; Daley, P. F., Raschke, K., Ball, J. T. and Berry, J. A., Plant Physiol. 90, 1233, 1989; Osmond, C. B., Berry, J. A., Balachandra, S., Buchen-Osmond, C., Daley, P. E, and Hodgson, R. A., Bot. Acta 103, 226, 1990). Moreover, fluorescence varies across the face of the leaf in ways diagnostic of leaf function (Daley et al., 1989).
At room temperature, PS.sub.II is the major and most variable source of fluorescence (Bolhar-Nordenkampf and Oquist, 1993). Fluorescence signals from PS.sub.II are sensitive to environmental stresses, such as high temperature, chilling, freezing, drought, and excess radiation.(Smillie, R. M., "The Useful Chloroplast: a New Approach for Investigating Chilling Stress in Plants," in Low Temperature Stress in Crop Plants, Lyons, J. M., Graham, D. and Raison, J. K. Eds., Academic Press, New York, 1979, p. 187; Havaux and Lannoye, 1984; Powles, 1984).
Although fluorescence reports directly on the light reaction, it can also be used to assay the dark reaction in plants with C-4 metabolism (Ning, L., Ozanich, R., Daley, L. S. and Callis, J. B., Spectroscopy 9(7), 41, 1994), such as corn (Zea mays L.). This is because in C-4 plants most electron flow through PS.sub.II is used, via PS.sub.I, to make NADPH for carbon fixation in the dark reaction. Fluorescence from PS.sub.II is thus closely correlated with the quantum yield of CO.sub.2 fixation (Edwards, G. E. and Baker, N. R., Photosynthesis Research 37, 89, 1993).
Thus, imaging of plant fluorescence could be a rich source of information on plant pathology and physiology, particularly for the agricultural industry. However, a need exists for simple methods of accessing and interpreting the information contained in plant fluorescence.
Quantum Yield
One simple method of obtaining information about plant pathology and physiology from plant fluorescence is to compute the quantum yield or maximum intrinsic efficiency, Y, of PS.sub.II, which is given by the ratio (Bolhar-Nordenkampf and Oquist, 1993): EQU Y=(F.sub.P -F.sub.O)/F.sub.P (1)
Here F.sub.P is the peak fluorescence and F.sub.O is the origin fluorescence in the Kautsky curve.
Unfortunately, Y is not a perfect measure of photochemical efficiency, because stress can lead to restrictions on electron flow or carbon fixation not manifest in F.sub.O or F.sub.P. Moreover, measurement of F.sub.O is difficult, because F.sub.O occurs over a very short time period. In addition, measurement of F.sub.P must be precisely timed to capture the peak fluorescence, a time that varies with plant pathology and physiology. Thus, a need exists for alternatives to Eq. 1 that will more conveniently and accurately measure photochemical efficiency.
Robotic Agriculture
Photosynthetic efficiency is of major economic interest because it is directly coupled to agricultural productivity. The agricultural industry attempts to maximize photosynthetic efficiency by providing plants with appropriately timed inputs, such as pesticides, fertilizer, and water.
Currently, agricultural pesticide, fertilizer, and irrigation schedules are determined by manual field scouting, in which a trained observer interprets leaf signals and other indicators of plant health. Field scouting, supplemented by laboratory analysis, is an expensive and time-consuming process. Moreover, following scouting, agricultural inputs are applied uniformly on entire fields, leading to the excess use and runoff of pesticides, fertilizer, and water.
As an alternative, robots could be used to supply an individual mix of agricultural inputs to each plant in every part of a field. Already, devices exist to locate and spray individual weeds. Much more could be done in the future: pesticides could be rapidly applied only to afflicted plants, killing pests before they spread, or fertilizer and water could be supplied only as required by each plant.
However, to provide inputs to individual plants, a robot must receive and process information on the condition of individual plants. Therefore, a need exists for simple data inputs to help robotic agriculture.
Futures Market
In the volatile agricultural futures market, information on crop conditions is used to help predict the future prices of agricultural commodities. Prices vary as conditions vary; for example, futures prices for citrus may rise following frost damage to the citrus crop. When crop information is delayed or incomplete, market instabilities can arise. Therefore, a need exists for rapid determination of crop conditions, particularly conditions relating to harvest size.
Biomolecular Electronics
Biomolecular electronics is the use of biological molecules in the construction of electronic devices. Photosynthetic components are potentially valuable components of biomolecular electronic devices because they are essentially microscopic photoelectronic devices. As photons are absorbed by a photosynthetic system, electron transfer reactions are initiated that result in a unidirectional, spatial separation of charges across the photosynthetic membrane. In essence, this separation can be regarded as electron movement through an insulating medium, controlled at the molecular level (Boxer, S. G., Stocker, J., Franzen, S., and Salafsky, J., "Re-engineering photosynthetic reaction centers," in Molecular Electronic Science and Technology, Aviram, A. Ed., Amer. Inst. Physics, New York, 1992, pp. 226-236).
Two potential electronic uses of photosynthetic systems are: a) coupling of chloroplast components to electronic systems (Greenbaum, E., J. Phys. Chem. 94, 6151, 1990; Greenbaum, E., J. Phys. Chem. 96, 514, 1992; Greenbaum, E., "Biomolecular electronics and applications," in Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Meyers, R. M. Ed., VCH Publishers Inc., New York, 1995, pp. 98-103.), and b) re-engineering of photosynthetic reaction systems for bioelectronic applications (Boxer et al., 1992). However, the quality of the biomolecular electronic device built around photosynthetic components will depend on the efficiency of the photosynthetic components. Because photosynthetic materials differ greatly in efficiency, a need exists for a method to screen photosynthetic systems for their efficiency before they can be effectively exploited in biomolecular electronics.